Axioimager a2 fluorescent microscope

Membrane integrity was assessed using the fluorescent probes SYTO 9 and propidium iodide (PI) (LIVE/DEAD BacLight Bacterial Viability Kit, Molecular Probes, United States). The test strain S. aureus 209P was grown to the mid-log phase in CAMHBc. The obtained culture was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the pellet was dispersed in 10 mM HEPES buffer supplemented with 0.5% glucose and 0.45 mM CaCl₂ to an optical density of 0.2 (OD₆₂₀) that corresponded to 10⁸ CFU/ml. Bacterial cells in 50 μl were transferred to a 96-well microtiter plate (Eppendorf, Germany). Measurements of SYTO 9 fluorescence kinetics were performed on a Fluoroskan Ascent FL plate reader (Thermo Fisher Scientific, United States) at 485 nm excitation and 535 nm emission wavelengths as described earlier (Rogozhin et al., 2018 (link)). Assessment of the viability of bacterial cells was performed by the agar-drop plate assay using one part of the treated bacterial population. Another part of the treated bacteria was used to prepare samples for fluorescence microscopy. Fluorescence microscopy was performed using a Zeiss Axio Imager A2 fluorescent microscope (Carl Zeiss, Germany) equipped with filter sets useful for simultaneous viewing of SYTO 9 and PI fluorescence.

The number of dendrites crossing each circle was counted manually. Studies of GluN2A knockdown on hippocampal primary hippocampal neurons were also carried out using Zeiss Axio Imager A2 fluorescent microscope (Zeiss, Jena, Germany) with Cool Snap EZ camera (Visitron System) and MetaMorph Imaging software (MDS Analytical Technologies). Up to 3 coverslips were treated individually and processed per group. The same exposure time and intensity were taken for each coverslip among the different groups. Upon background subtraction using Fiji software, images of fluorescent positive puncta were measured along secondary dendrites, right after the branching point. The synaptic immunofluorescence intensities were assessed in a region of 400 nm × 400 nm square set by the mask in the channel for Homer1, a synaptic marker. The mask was created semi-automatically using Openview software. The intensities of the puncta positive for GluN2A were measured at points of co-localization with Homer1; values were normalized to mean of the control and plotted.

Image analysis was carried out using a Zeiss Axio Imager A2 fluorescent microscope (Zeiss, Jena, Germany) with Cool Snap EZ camera (Visitron System, Puchheim, Germany) and MetaMorph Imaging software (MDS Analytical Technologies, Ismaning, Germany). Up to 3 coverslips were treated individually and processed per group. For each coverslip, the same exposure time and intensity were taken among the different groups. After background subtraction, the fluorescence intensity of the immunosignal was measured along dendrites right after the first branching point using ImageJ software. The synaptic immunofluorescence intensities of pan-AKT and phospho-AKT were assessed in a region of 400 nm × 400 nm square set by the mask generated based on synaptic marker Shank3. The Shank3 mask was created semi-automatically using OpenView software [34 (link)].